This invention relates to novel peptide fragments of a blood coagulation inhibitor known as tissue factor inhibitor (TFI) but preferably as lipoprotein-associated coagulation inhibitor (LACI). More particularly, the invention relates to a method of inhibiting Factor Xa or Factor VIIa/TF complex with novel peptide fragments of LACI.
The coagulation cascade that occurs in mammalian blood comprises two distinct systems--the so-called intrinsic and extrinsic systems. The latter system is activated by exposure of blood to tissue thromboplastin (Factor III), hereinafter referred to as tissue factor (TF). Tissue factor is a lipoprotein that arises in the plasma membrane of many cell types and in which the brain and lung are particularly rich. Upon coming into contact with TF, plasma Factor VII or its activated form, Factor VII.sub.a, forms a calcium-dependent complex with TF and then proteolytically activates Factor X to Factor X.sub.a, and Factor IX to Factor IX.sub.a, thereby triggering a cascade of events which leads eventually to the formation of thrombin and a fibrin clot.
In copending application Ser. No. 07/77,366, filed July 23, 1987, a purified tissue factor inhibitor (TFI) is disclosed which was secreted from HepG2 cells. It was found to exist in two forms, a TFI.sub.1, migrating at about 37-40,000 daltons and a TFI.sub.2 at about 25-26,000 daltons, as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). A partial N-terminal amino acid sequence for the TFI was assigned as: ##STR1## wherein X--X had not been determined. The disclosure of said application and the related disclosure by Broze and Miletich, Proc. Natl. Acad. Sci. USA 84, 1886-1990 (1987) are incorporated herein by reference.
In copending application Ser. No. 07/123,753, filed Nov. 23, 1987, the complete coding sequence of a cDNA clone representing essentially the full size TFI or LACI is disclosed. The cDNA sequence encoded a 31,950 Dalton protein of 276 amino acids which included 18 cysteines and 7 methionines. The translated amino acid sequence showed that a signal peptide of about 28 amino acids preceded the mature LACI protein. The "mature" LACI was defined to include both LACI and methionyl LACI by virtue of the ATG translational codon in the .lambda.P9 clone described therein.
There are three potential N-linked glycosylation sites in the LACI protein with the sequence Asn-X-Ser/Thr, wherein X can be any of the common 20 amino acids. These sites are at amino acid positions Asn 145, Asn 195, and Asn 256, when the first methionine after the 5'-noncoding region is assigned amino acid position +1.
The translated amino acid sequence of LACI showed several discernible domains, including a highly negatively charged N-terminal, a highly positively charged carboxy-terminal, and an intervening portion consisting of 3 homologous domains with sequences typical of Kunitz-type enzyme inhibitors. Based on a homology study, LACI appeared to be a member of the basic protease inhibitor gene superfamily.
In said copending application Ser. No. 07/123,753, the three tandemly repeated Kunitz-type serine protease inhibitory domains were shown to exist at LACI amino acid residues 47-117 (1), 118-188 (2) and 210-280 (3), respectively, based on the above amino acid numbering system. The disclosure of said application and the related disclosure by Wun et al., J. Biol. Chem. 263 (13), 6001-6004 (1988) are incorporated herein by reference.